Use of GPR30 receptor ligands to mediate neurotrophic action

ABSTRACT

Neurological dysfunction is prevented or treated by the administration of ligands that activate the GPR30 receptor. Ligands include estrogens and structurally related molecules. Preferably the GPR30 ligand is an orally available drug that can cross into the brain from blood. Screening methods are provided for ligands that do not activate other estrogen receptors, and therefore do not have the classical estrogenic effects attributable to these receptors.

CROSS REFERENCE

This application is a divisional of earlier filed application Ser. No.09/452,531 filed Dec. 1, 1999 now U.S. Pat. No. 6,265,147 to whichapplication is claimed priority and which application is incorporatedherein by reference in its entirety.

INTRODUCTION

Neurodegenerative diseases are characterized by the dysfunction anddeath of neurons, leading to the loss of neurologic functions mediatedby the brain, spinal cord and the peripheral nervous system. Thesedisorders have a major impact on society. For example, approximately 4to 5 million Americans are afflicted with the chronic neurodegenerativedisease known as Alzheimer's disease. Other examples of chronicneurodegenerative diseases include diabetic peripheral neuropathy,multiple sclerosis, amyotrophic lateral sclerosis, Huntington's diseaseand Parkinson's disease. Normal brain aging is also associated with lossof normal neuronal function and may entail the depletion of certainneurons. Not all neurodegenerative diseases are chronic. Stroke is anacute neurodegenerative disease. Sudden loss of neurons may alsocharacterize the brains of patients with epilepsy and those that sufferhypoglycemic insults and traumatic injury of the brain, peripheralnerves or spinal cord.

Though the mechanisms responsible for the dysfunction and death ofneurons in neurodegenerative disorders are not well understood, a commontheme is that loss of neurons results in both the loss of normalfunctions and the onset of adverse behavioral symptoms. For example,patients with Alzheimer's disease demonstrate memory loss and cognitivedeficits as well as bizarre and sometimes aggressive behaviors.Therapeutic agents that have been developed to retard loss of neuronalactivity and survival are largely ineffective. Some have toxic sideeffects that limit their usefulness. Other promising therapies, such asneurotrophic factors, are prevented from reaching their target sitebecause of their inability to cross the blood-brain barrier. Theblood-brain barrier is a complex of structural and enzymatic componentsthat retards the passage of both large and charged small molecules,thereby limiting the access of such molecules to cells in the brain.

Senile dementia of the Alzheimer's type is a debilitatingneurodegenerative disease, mainly afflicting the elderly; it ischaracterized by a progressive intellectual and personality decline, aswell as a loss of memory, perception, reasoning, orientation andjudgment. One highly reproducible feature of the disease is dysfunctionand loss of selected populations of neurons in the brain. Among theseare cholinergic neurons of the basal forebrain, whose normal functioncontributes importantly to attention, learning and memory. There is anobserved decline in the function of cholinergic systems, and a severedepletion of these cholinergic neurons.

It is increasingly apparent that estrogen has an important role inregulating neuronal survival and function. There is perhaps no betterdemonstration of this than the finding that administration of estrogento postmenopausal women significantly decreases the incidence ofAlzheimer's disease. These striking data have encouraged new interest inthe biological functions of estrogen and, in particular, of its actionson neurons.

In recent years it has become apparent that estrogen enhances thedifferentiation of neurons, including the outgrowth of processes fromCNS neurons; estrogen binds to specific receptors on neurons in thebrain and regulates the levels of classical neurotrophic factors,including nerve growth factor (NGF) and brain-derived neurotrophicfactor (BNDF), and their receptors. These findings are evidence thatestrogen is itself a neurotrophic factor, and they argue strongly thatestrogen's neurotrophic actions are responsible for enhancing thesurvival and function of neurons important for memory and learning inpostmenopausal women. Indeed, these data have suggested that estrogen'sneurotrophic actions could benefit the function and survival of neuronsin both women and men and that estrogen, or an estrogen derivative thatis active on estrogen receptors, could be used to treat patients withdisorders in which there is dysfunction and death of neurons.

Estrogen elicits a selective enhancement of the growth anddifferentiation of axons and dendrites (neurites) in the developingbrain. Widespread colocalization of the receptors for estrogen andneurotrophic factors in a number of neuronal populations, includingneurons of the basal forebrain, cerebral cortex, sensory ganglia, andPC12 cells, has been correlated with the differential and reciprocaltranscriptional regulation of these receptors by their ligands. Evenmore important, estrogen and neurotrophic factor receptor coexpressionleads to convergence or cross-coupling of their signaling pathways,particularly at the level of the mitogen-activated protein (MAP) kinasecascade. The ability of estrogen to regulate a broad array ofcytoskeletal and growth-associated genes involved in neurite growth anddifferentiation is of great interest in the development ofpharmaceutical agents and methodologies for the treatment ofneurological disorders.

Relevant Literature

Estrogen elicits a selective enhancement of the growth anddifferentiation of axons and dendrites (neurites) in the developing CNS.There is widespread colocalization of estrogen and neurotrophinreceptors within developing forebrain neurons and reciprocaltranscriptional regulation of these receptors by their ligands(Toran-Allerand (1996) Dev Neurosci 18(1-2):36-48). Estradiol elicitedrapid tyrosine phosphorylation/activation of mitogen-activated protein(MAP) kinases. This extracellular signal-regulated protein kinaseactivation was inhibited successfully by the MEK1 inhibitor PD98059, butnot by the estrogen receptor (ER) antagonist ICI 182,780, and did notappear to result from estradiol-induced activation of trk (Singh et al.(1999) J Neurosci 19(4):1179-88).

U.S. Pat. No. 5,843,934, issued Dec. 1, 1998, a method is described forconferring a cytoprotective effect on a population of cells byadministering an estrogen compound having insubstantial sex relatedactivity.

SUMMARY OF THE INVENTION

Compositions and methods are provided for the treatment of neurologicaldysfunction, by the administration of ligands that activate the GPR30receptor. Ligands include, but are not limited to, estrogens andstructurally related molecules. Conditions that benefit from protectionof neurons include brain trauma, stroke, multiple sclerosis,neurodevelopmental disorders and neurodegenerative disorders including,but not limited to, Alzheimer's disease, Parkinson's disease, andamyotrophic lateral sclerosis.

In a preferred embodiment, the GPR30 ligand is an orally available drugthat can cross into the brain from blood. Of particular interest areestrogen derivatives that do not activate other estrogen receptors, andtherefore do not have the classical estrogenic effects attributable tothese receptors.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A and 1B: Total poly(A)-mRNAs were extracted from PC12 cells, anER- and GPR30-positive control cell line (+) and a GPR30-negative cellline (−). The mRNAs were separated, transferred and blotted with³²P-labeled probes specific for ERs (A) and GPR30 (B). The Northernblots show that, PC12 cells express no detectable ERs but a low level ofGPR30 message. Expression of GPR30 in PC12 cells is further confirmed byRT-PCR using GPR30 specific primers. The results are presented in thelower panel of B.

FIGS. 2A, 2B, 2C, 2D: 17β-estradiol elicits Rap1, but not Ras,activation in PC12 cells. Serum-starved PC12 cells were treated with 100nM 17β-estradiol for the indicated time intervals. Activated Ras (A) andRap1 (B) were precipitated from cell lysates and analyzed as describedin the Materials and Methods. The results show that 17β-estradiolinduces Rap1 activation (B) and causes no detectable Ras activation (A).Rap1 activation induced by 100 nM 17β-estradiol can be blocked by 1 μMtomaxifen (C). Tomaxifen itself has no effect on Rap1 activation in PC12cells D.

FIG. 3: 17β-estradiol induces prolonged Erk activation in PC12 cells. Asin FIG. 2, serum-starved PC12 cells were treated with 100 nM17β-estraldiol for the indicated time intervals. Cells were-rinsed andlysed in RIPA buffer. Proteins were separated on SDS-PAGE, blotted ontoPVDF membrane. The blot was probed for activated Erks (i.e activated MAPkinase) using rabbit IgGs against activated Erk kinases (UBI).

FIGS. 4A and 4B: 17β-estradiol induces PC12 cell differentiation. PC12cells were maintained in either serum-free media (A) or serum-free mediasupplemented with 100 nM 17β-estradiol (B) for 6 days. Cells weremonitored with phase-contrast microscope and typical images werecaptured using a CCD camera. Differentiated PC12 cells (arrows) can beobserved with 17β-estradiol treatment (B), whereas massive cell deathoccurred in the control (A).

FIG. 5: Induction of the 145 kDa intermediate neurofilament expressionby 17β-estradioi in PC12 cells. PC12 cells were maintained in eitherserum-free media supplemented with either NGF or 17β-estradiol at theindicated concentrations for 3 days. Treated as well as control cellswere lysed in RIPA buffer and the lysates were separated on SDS-PAGE,transferred onto PVDF membrane. The blot was probed with specificantibodies against the 145 kDa intermediate neurofilament.

DETAILED DESCRIPTION OF THE INVENTION

Neurological disorders are treated by administering a therapeutic doseof a ligand to the GPR30 receptor. The GPR30 receptor is expressed inneural cells, and when activated can provide for neuroprotection fromtrauma and disease. While GPR30 can be activated by estrogens, it isdistinct from the classical estrogen receptors. Of particular interestare estrogen derivatives that do not activate other estrogen receptors,and therefore do not have the classical estrogenic effects attributableto these receptors. In a preferred embodiment, the GPR30 ligand is anorally available drug that can cross into the brain from blood.

The subject methods are used for prophylactic or therapeutic purposes.As used herein, the term “treating” is used to refer to both preventionof disease, and treatment of pre-existing conditions. The prevention ofdisease is accomplished by administration of the GPR30 ligands prior todevelopment of overt disease. The treatment of ongoing disease, in orderto stabilize or improve the clinical symptoms of the patient, is ofparticular interest. Such treatment is desirably performed prior tocomplete loss of function in the affected tissues.

The GPR30 receptor is also used in screening assays to determine ligandsthat are suitable for use in the neuroprotective methods. Test compoundsare screened for those that have the desired properties, through theirspecific binding and activation of GPR30, which preferably lack estrogenaction at classical estrogen response elements. Compounds of interestfor screening include, without limitation, combinatorial libraries ofsteroid and steroid derivatives; targeted modifications of estrogeniccompounds; environmental compounds, which can be derived from a widevariety of sources including plants, soil, water, foods; syntheticcompounds such as chlorinated organics, polycyclic aromatichydrocarbons, herbicides; pesticides; pharmaceuticals; and the like.

Definitions

It is to be understood that this invention is not limited to theparticular methodology, protocols, cell lines, animal species or genera,constructs, and reagents described, as such may vary. It is also to beunderstood that the terminology used herein is for the purpose ofdescribing particular embodiments only, and is not intended to limit thescope of the present invention which will be limited only by theappended claims.

“Neurologic disorder” is defined here and in the claims as a disorder inwhich dysfunction and loss of neurons occurs either in the peripheralnervous system or in the central nervous system. Examples ofneurodegenerative disorders include: chronic neurodegenerative diseasessuch as Alzheimer's disease, Parkinson's disease, Huntington's chorea,diabetic peripheral neuropathy, multiple sclerosis, amyotrophic lateralsclerosis; aging; and acute neurodegenerative disorders including:stroke, traumatic brain injury, peripheral nerve damage, hypoglycemia,spinal cord injury, epilepsy, and anoxia and hypoxia.

GPR30: The G protein coupled receptor, GPR30 is described in the art,for example see Owman et al. (1996) Biochem. Biophys. Res. Commun.228:285-292; or Carmeci et al. (1997) Genomics 45:607-617. GPR30 wasdiscovered as a result of its increased expression in ER-positive breastcarcinoma cell lines, but not in ER-negative breast carcinoma celllines. Overexpression of GRP30 confers increased estrogen responsivityto cells derived from breast cancer tissue. GPR30 is expressed in thebrain (Carmeci et al. (1997) Genomics 45:607-617). It is structurallydistinct from all other known estrogen receptors.

The nucleotide sequence of naturally occurring human GPR30 may beaccessed in Genbank, accession number Y08162. Those of skill in the artcan easily use the nucleotide sequences to produce the coding sequence,to genetically modify cells to express the protein, or to produce thepurified GPR30 polypeptide for screening and other assays.

The nucleic acids of the invention can be introduced into suitable hostcells using a variety of techniques which are available in the art, suchas transferring polycation-mediated DNA transfer, transfection withnaked or encapsulated nucleic acids, liposome-mediated DNA transfer,intracellular transportation of DNA-coated latex beads, protoplastfusion, viral infection, electroporation, gene gun, calciumphosphate-mediated transfection, and the like. The nucleic acids can beprovided as a linear molecule or within a circular molecule. They can beprovided within autonomously replicating molecules (vectors) or withinmolecules without replication sequences. They can be regulated by theirown or by other regulatory sequences, as is known in the art.

The nucleic acid encoding GPR30 are used to express a partial orcomplete gene product. Constructs can be generated by recombinantmethods, synthetically, or in a single-step assembly of a gene andentire plasmid from large numbers of oligodeoxyribonucleotides isdescribed by, e.g. Stemmer et al., Gene (Amsterdam) (1995) 164(1):49-53.The choice of appropriate vector is well within the skill of the art.Many such vectors are available commercially.

Appropriate nucleic acid constructs are purified using standardrecombinant DNA techniques as described in, for example, Sambrook etal., Molecular Cloning: A Laboratory Manual, 2nd Ed., (1989) Cold SpringHarbor Press, Cold Spring Harbor, N.Y. The gene product encoded by anucleic acid of the invention is expressed in any expression system,including mammalian cells, and particularly human neural cells.

The coding sequences may be linked to regulatory sequences asappropriate to obtain the desired expression properties. These caninclude promoters attached either at the 5′ end of the sense strand orat the 3′ end of the antisense strand, enhancers, terminators,operators, repressors, and inducers. The promoters can be regulated orconstitutive. In some situations it may be desirable to useconditionally active promoters, such as tissue-specific or developmentalstage-specific promoters. These are linked to the desired nucleotidesequence using the techniques described above for linkage to vectors.Any techniques known in the art can be used.

GPR30 Ligands: Ligands to the GPR30 are molecules that specifically bindto the GPR30 receptor. Such ligands can include low molecular weightnative molecules, such as estrogens, as well as synthetic derivativecompounds such as diethylstilbesterol. Ligands of particular interestare able to bind to GPR30 protein to create a ligand/receptor complex.GPR30 is a G-protein coupled receptor whose overall structure isdistinct from that of all other known estrogen receptors and, indeedform the structural class into which these other receptors fall. Whilesome of the ligands for GPR30 are shared with these other receptors,others are expected to be distinct, binding only to GPR30. A compoundthat binds a steroid receptor and mimics the effects of the nativeligand may be referred to as an “agonist”, while a compound thatinhibits this effect is called an “antagonist.”

The effectiveness of known modulators of steroid receptors is oftentempered by their undesired side-effect profile, particularly duringlong-term administration. For example, the effectiveness ofdiethylstilbesterol respectively, as female birth control agents must beweighed against the increased risk of breast cancer and heart disease towomen taking such agents. Accordingly, GPR30 ligands of interest havegood specificity for binding GPR30 receptors, but which have reduced orno cross-reactivity for other steroid or intracellular receptors.

Estrogens: is a generic term used to refer to a class of steroid ornon-steroid estrogenic hormones. Various substances including not onlynatural substances, but also synthetic substances, are known asestrogens (Environmental Health Perceptives (1985) Vol. 61, pp. 97-110).Various assays may be used to determine whether a compound hasestrogenic activity, for example, cellular assays as described in Webbet al. (1993) Mol. Endocrinol. 6:157-167.

Frequently the term estrogen is used to refer to the three naturallyproduced estrogens that are present in significant quantities in theplasma of human females: 17β-estradiol, estrone, and estriol. Thesecompounds have the following chemical structures.

In mammalian cells, the estrogens 17β-estradiol and estrone arecontinuously interconverted by 17β-oxidoreductase, and are generallymetabolized via two major pathways: hydroxylation at the 16α-position orhydroxylation at the 2- or 4-position by cytochrome P-450. The latterpathway produces catechol estrogens.

Other estrogen compounds included in this definition are estrogenderivatives, estrogen metabolites and estrogen precursors as well asthose molecules capable of binding cell associated estrogen receptor aswell as other molecules where the result of binding specificallytriggers a characterized estrogen effect. Also included are mixtures ofmore than 1 estrogen.

Of particular interest for the subject methods are estrogen derivatives,a great many of which have been described in the art. The extensiveliterature on steroid chemistry provides a wide variety of compounds foruse in the subject screening and therapeutic methods. To provide only asmall example, some known derivatives of estrogens includealpha-estradiol dipropionate, estradiol mustard, polyestradiolphosphate, estradiol 3-[bis(2-chloroethyl)carbamate], estradiolcyclopentylpropionate, beta-estradiol diacetate, estradiol undecylate,2-hydroxyestradiol, beta-estradiol-3-benzoate, cloxestradiol, 6β-hydroxyestradiol-17β, 17-ethynyl estradiol, estradiol,4-hydroxyestradiol, ethynylestradiol 3-methyl ether,estradiol-17-valerate, estradiol-17-caprylate,estradiol-17α3-d-glucuronoside, 17α-(n-acetyl-d-glucosaminyl)-estradiol3-d-glucuronoside, 2-methoxyestradiol-17β,estradiol 17-octadecanoate,almestrone, neo-oestronol ii, nitroestrone, estrone benzoate,2-hydroxyestrone, sodium estrone sulfate, estrone-3-sulfate,stilbestrone, neo-estrone, quingestrone, anhydrohydroxynorprogestrone,pentagestrone, metigestrona, piperazine estrone sulfate, fluoxymestrone,westron, edogestrone, 16α-hydroxyestrone, 2-methoxyestrone,estrone-2,4,16,16-d4, estrone-16,16-d2, estrone-2,4-d2, estriolpropionate, estriol acetate benzoate, nilestriol, estriol succinate,epiestriol, 3-methoxy-17-epiestriol; and the like as known in the art.

Estrogen receptor: as used herein, is intended to mean the “classical”estrogen receptors. The molecules are also known as ESR1 (estrogenreceptor α) and ESR2 (estrogen receptor β). The sequences of theseproteins and corresponding nucleic acids are well known in the art. ESR1is a member of the superfamily of nuclear receptors, which can transduceextracellular signals into transcriptional responses. The nucleotidesequence encoding ESR1 was described by Green et al. (1986) Nature320:134-139, 1986; and Green et al. (1986) Science 231:1150-1154. Thesequences may be accessed at Genbank, accession number NM_(—)000125.

ESR2 is homologous to ESR1 and has an overlapping but nonidenticaltissue distribution. The DNA-binding domain of ESR-β is 96% conservedcompared to ESR1, and the ligand-binding domain shows 58% conservedresidues (Mosselman et al. (1996) FEBS Lett. 392:49-53). The sequencemay be accessed at Genbank, accession number AF051427.

In preferred embodiments of the invention, the methods utilize GPR30ligands that do not substantially activate ESR1 or ESR2. Therefore, inscreening compounds, one may determine two attributes for a candidatecompound: whether it activates GPR30, and whether it lacks activation ofESR1 and/or ESR2. Activation of ESR1 and ESR2 by hormone involves atleast two steps. First, hormone binding initially relieves repression, aproperty imposed on ER in cis by its ligand-binding domain (EBD).Subsequently, the derepressed ER binds specific genomic sites andregulates transcription. In addition to the natural hormone, ER binds abroad range of ligands that evoke a spectrum of responses ranging fromfull ER activation by agonists to partial activation and inhibition bypartial or complete antagonists.

A number of assays have been described in the literature for determiningwhether a candidate compound has activity on ERs. For example, Logie etal. (1998) Mol Endocrinol 12(8):1120-32 describe a nontranscriptionalassay for responsiveness based on Flp recombinase/human EBD proteinchimeras. These fusion proteins transduce the transient event of ligandbinding into a permanent DNA change in a human cell line system.Agonists and antagonists can be functionally distinguished in anontranscriptional assay. The ability of a cell line to activate atransfected estrogen response element (ERE)-containing reporter gene isassessed by Zhang et al. (1999) Mol Endocrinol 13(4):632-43. Competitionbinding analysis for estrogen receptors are well known, e.g. seeHanstein et al. (1999) Mol Endocrinol 13(1):129-37.

U.S. Pat. No. 5,723,291, Kushner et al. describes methods for screeningcompounds with activity at ERs. The assay use cells comprising promotershaving an AP1 site linked to a reporter gene. Compounds capable ofinducing or blocking expression of the reporter gene can thus beidentified. Jorgensen et al. (1998) APMIS 106(1):245-51 reviewscombinations of assay methods, such as direct in vitro measurement ofinteraction between a potential estrogenic chemical and the ER withmethods that are based on mammalian cells or whole animals, or to assaygene expression directly by methods such as differential display, wherethe expression of both genes known to be regulated directly by thereceptor and genes regulated by other pathways can be monitored.

Drug Screening Assays: These are used to identify GPR30 ligands thatbind to and activate the GPR30 receptor. Drug screening identifiesagents that enhance GPR30 activity. Of particular interest are screeningassays for agents that have a low toxicity for human cells. In vitro orin vivo binding assays can be used to assess the extent to which acompound behaves as a specific ligand. Other assays are available totest for the activation of signaling pathways by ligand binding. Oneassay based on the finding that GPR30 activation results in theactivation of the small GTPase Rap1, and of the other enzymes in the MAPkinase signaling pathway that is activated by Rap1. The assay forevaluating the activation of Rap1 examines the extent to which Rap1 isbound to GTP and employs a method as described by Herrmann et al. (1995)J. Biol. Chem 270:2901-2905. A wide variety of assays are available toassess the activation status of the enzymes downstream from Rap1. Theseinclude in vitro kinase assays for B-Raf, MEK, MAP kinase and Rsk(Cowley et al. (1994) Cell 77:841-852; Vossler et al. (1997) Cell89:73-82). The activation of these proteins and some of their downstreamtargets (e.g. Elk-1 and CREB) may also be assessed through the use ofantibodies to phosphorylated tyrosine or serine or threonine residues,which marks their active state (Payne et al. (1991) EMBO J.10(4):885-892).

The GPR30 protein may also be used for determination ofthree-dimensional crystal structure, which can be used for modelingintermolecular interactions, including those that characterize thebinding of a ligand to GPR30. One may examine the interactions ofligands with receptors, as reviewed by Borman (1992) Chem. Eng. News70:18-26. Drug design may include studies which focus on the binding ofa ligand to a protein, including assays for the detection of ligandbinding.

The term “agent” as used herein describes any molecule, e.g. smallorganic molecule, with the capability of binding to, and activatingGPR30. Generally a plurality of assay mixtures are run in parallel withdifferent agent concentrations to obtain a differential response to thevarious concentrations. Typically, one of these concentrations serves asa negative control, i.e. at zero concentration or below the level ofdetection.

Candidate agents encompass numerous chemical classes, though typicallythey are organic molecules, preferably small organic compounds having amolecular weight of more than 50 and less than about 2,500 daltons.Candidate agents comprise functional groups necessary for structuralinteraction with proteins, particularly hydrogen bonding, and typicallyinclude at least an amine, carbonyl, hydroxyl or carboxyl group,preferably at least two of the functional chemical groups. The candidateagents often comprise cyclical carbon or heterocyclic structures and/oraromatic or polyaromatic structures substituted with one or more of theabove functional groups.

For example, candidate agents may utilize the structures and methodsdescribed by Fink et al. (1999) Chem. Biol. 6:205-219, of an estrogenpharmacophore that consists of a simple heterocyclic core scaffold,amenable to construction by combinatorial methods, onto which areappended 3-4 peripheral substituents that embody substructural motifscommonly found in nonsteroidal estrogens. Members of the imidazole,thiazole or isoxazole classes generally have weak binding for ESR1, andare well suited for combinatorial synthesis using solid-phase methods.

Candidate agents may be obtained from a wide variety of sourcesincluding libraries of synthetic or natural compounds. For example,numerous means are available for random and directed synthesis of a widevariety of organic compounds and biomolecules, including expression ofrandomized oligonucleotides and oligopeptides. Alternatively, librariesof natural compounds in the form of bacterial, fungal, plant and animalextracts are available or readily produced. Additionally, natural orsynthetically produced libraries and compounds are readily modifiedthrough conventional chemical, physical and biochemical means, and maybe used to produce combinatorial libraries. Known pharmacological agentsmay be subjected to directed or random chemical modifications, such asacylation, alkylation, esterification, amidification, etc. to producestructural analogs.

Where the screening assay is a binding assay, one or more of themolecules may be joined to a label, where the label can directly orindirectly provide a detectable signal. Various labels includeradioisotopes, fluorescers, chemiluminescers, enzymes, specific bindingmolecules, particles, e.g. magnetic particles, and the like. Specificbinding molecules include pairs, such as biotin and streptavidin,digoxin and antidigoxin etc. For the specific binding members, thecomplementary member would normally be labeled with a molecule thatprovides for detection, in accordance with known procedures.

A variety of other reagents may be included in the screening assay.These include reagents like salts, neutral proteins, e.g. albumin,detergents, etc that are used to facilitate optimal protein-proteinbinding and/or reduce non-specific or background interactions. Reagentsthat improve the efficiency of the assay, such as protease inhibitors,nuclease inhibitors, anti-microbial agents, etc. may be used. Themixture of components are added in any order that provides for therequisite binding. Incubations are performed at any suitabletemperature, typically between 4 and 40° C. Incubation periods areselected for optimum activity, but may also be optimized to facilitaterapid high-throughput screening. Typically between 0.1 and 1 hours willbe sufficient.

Pharmaceutical Formulation: The GPR30 ligand may be combined with apharmaceutically acceptable carrier, which term includes any and allsolvents, dispersion media, coatings, anti-oxidant, isotonic andabsorption delaying agents and the like. The use of such media andagents for pharmaceutically active substances is well known in the art.Except insofar as any conventional media or agent is incompatible withthe active ingredient, its use in the therapeutic compositions andmethods described herein is contemplated. Supplementary activeingredients can also be incorporated into the compositions.

The formulation may be prepared for use in various methods foradministration. The formulation may be given orally, by inhalation, ormay be injected, e.g. intravascular, intratumor, subcutaneous,intraperitoneal, intramuscular, etc.

The dosage of the therapeutic formulation will vary widely, dependingupon the nature of the disease, the frequency of administration, themanner of administration, the clearance of the agent from the host, andthe like. The initial dose may be larger, followed by smallermaintenance doses. The dose may be administered as infrequently asweekly or biweekly, or fractionated into smaller doses and administereddaily, semi-weekly, etc. to maintain an effective dosage level. In somecases, oral administration will require a higher dose than ifadministered intravenously.

The GPR30 ligand of the invention can be incorporated into a variety offormulations for therapeutic administration. More particularly, thecomplexes can be formulated into pharmaceutical compositions bycombination with appropriate, pharmaceutically acceptable carriers ordiluents, and may be formulated into preparations in solid, semi-solid,liquid or gaseous forms, such as tablets, capsules, powders, granules,ointments, solutions, suppositories, injections, inhalants, gels,microspheres, and aerosols. As such, administration of the GPR30 ligandcan be achieved in various ways. The GPR30 ligand may be systemic afteradministration or may be localized by the use of an implant that acts toretain the active dose at the site of implantation.

The following methods and excipients are merely exemplary and are in noway limiting. For oral preparations, the GPR30 ligand can be used aloneor in combination with appropriate additives to make tablets, powders,granules or capsules, for example, with conventional additives, such aslactose, mannitol, corn starch or potato starch; with binders, such ascrystalline cellulose, cellulose derivatives, acacia, corn starch orgelatins; with disintegrators, such as corn starch, potato starch orsodium carboxymethylcellulose; with lubricants, such as talc ormagnesium stearate; and if desired, with diluents, buffering agents,moistening agents, preservatives and flavoring agents.

The GPR30 ligand can be formulated into preparations for injections bydissolving, suspending or emulsifying them in an aqueous or nonaqueoussolvent, such as vegetable or other similar oils, synthetic aliphaticacid glycerides, esters of higher aliphatic acids or propylene glycol;and if desired, with conventional additives such as solubilizers,isotonic agents, suspending agents, emulsifying agents, stabilizers andpreservatives.

The GPR30 ligand can be utilized in aerosol formulation to beadministered via inhalation. The compounds of the present invention canbe formulated into pressurized acceptable propellants such asdichlorodifluoromethane, propane, nitrogen and the like.

Furthermore, the GPR30 ligand can be made into suppositories by mixingwith a variety of bases such as emulsifying bases or water-solublebases. The GPR30 ligand of the present invention can be administeredrectally via a suppository. The suppository can include vehicles such ascocoa butter, carbowaxes and polyethylene glycols, which melt at bodytemperature, yet are solidified at room temperature.

Implants for sustained release formulations are well-known in the art.Implants are formulated as microspheres, slabs, etc. with biodegradableor non-biodegradable polymers. For example, polymers of lactic acidand/or glycolic acid form an erodible polymer that is well-tolerated bythe host. The implant containing GPR30 ligand is placed in proximity tothe site of action, so that the local concentration of active agent isincreased relative to the rest of the body.

Unit dosage forms for oral or rectal administration such as syrups,elixirs, and suspensions may be provided wherein each dosage unit, forexample, teaspoonful, tablespoonful, gel capsule, tablet or suppository,contains a predetermined amount of the compositions of the presentinvention. Similarly, unit dosage forms for injection or intravenousadministration may comprise the compound of the present invention in acomposition as a solution in sterile water, normal saline or anotherpharmaceutically acceptable carrier. The specifications for the novelunit dosage forms of the present invention depend on the particularcompound employed and the effect to be achieved, and thepharmacodynamics associated with each active agent in the host.

The pharmaceutically acceptable excipients, such as vehicles, adjuvants,carriers or diluents, are readily available to the public. Moreover,pharmaceutically acceptable auxiliary substances, such as pH adjustingand buffering agents, tonicity adjusting agents, stabilizers, wettingagents and the like, are readily available to the public.

Methods of Use

A therapeutic dose of a GPR30 ligand is administered to a host sufferingfrom a neurologic disorder. Administration may be topical, localized orsystemic, depending on the specific disease. The compounds areadministered at an effective dosage that over a suitable period of timesubstantially arrests the disease progression. It is contemplated thatthe composition will be obtained and used under the guidance of aphysician for in vivo use.

The dose will vary depending on the specific GPR30 ligand utilized, typeof disorder, patient status, etc., at a dose sufficient to substantiallyprotect the neural cells from damage, dysfunction or death, whileminimizing side effects. Treatment may be for short periods of time,e.g. after trauma, or for extended periods of time, e.g. in theprevention or treatment of Alzheimer's disease.

It is to be understood that this invention is not limited to theparticular methodology, protocols, cell lines, animal species or genera,and reagents described, as such may vary. It is also to be understoodthat the terminology used herein is for the purpose of describingparticular embodiments only, and is not intended to limit the scope ofthe present invention which will be limited only by the appended claims.

As used herein the singular forms “a”, “and”, and “the” include pluralreferents unless the context clearly dictates otherwise. Thus, forexample, reference to “a cell” includes a plurality of such cells andreference to “the array” includes reference to one or more arrays andequivalents thereof known to those skilled in the art, and so forth. Alltechnical and scientific terms used herein have the same meaning ascommonly understood to one of ordinary skill in the art to which thisinvention belongs unless clearly indicated otherwise.

All publications mentioned herein are incorporated herein by referencefor the purpose of describing and disclosing, for example, the celllines, constructs, and methodologies that are described in thepublications which might be used in connection with the presentlydescribed invention. The publications discussed above and throughout thetext are provided solely for their disclosure prior to the filing dateof the present application. Nothing herein is to be construed as anadmission that the inventors are not entitled to antedate suchdisclosure by virtue of prior invention.

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how tomake and use the subject invention, and are not intended to limit thescope of what is regarded as the invention. Efforts have been made toensure accuracy with respect to the numbers used (e.g. amounts,temperature, concentrations, etc.) but some experimental errors anddeviations should be allowed for. Unless otherwise indicated, parts areparts by weight, molecular weight is average molecular weight,temperature is in degrees centigrade; and pressure is at or nearatmospheric.

Experimental

The reagents used in these studies were those specified in the originalreferences to methods employed, unless otherwise specified. We usedestablished cell culture methods for growing cells and for examiningtheir morphological responses (Zhou et al. (1994) Proc. Natl. Acad. SciUSA 91(9):3824-3828). To examine gene expression for GPR30 and theclassical ERs, standard methods were used to isolate mRNA and to analyzeit by Northern blot and by RT-PCR (GIBCO-BRL, Bethesda, Md.). Theactivity of Rap1 and of Ras were assayed using pull-down assays withfusion proteins that bind specifically to the GTP-bound forms of theseproteins, as described (Herrmann et al. (1995) J. Biol. Chem.270:2901-2905). These fusion proteins were produced essentially asdescribed by these authors. The activity of MAP kinase was determined byimmunoblotting cell lysates with antibodies to phosphorylated MAP kinase(Payne et al. (1991) EMBO J. 10(4):885-892). The source of theseantibodies was from New England Biolabs, Inc. (Beverley, Mass.).

The goal of these experiments was to ask if estrogen would activateGPR30 and induce the activation of signaling pathways known to beimportant for the survival and function of neurons. In short, to testthe idea that estrogen acted through GPR30 to act as a neurotrophic andneuroprotective factor.

NGF is a polypeptide neurotrophic factor that acts to enhance thefunction and survival of certain neurons in both the central andperipheral nervous system (Yuen and Mobley (1996) Ann Neurol.40(3):346-354). The ability of NGF to cause the differentiation ofneuronal cells and to increase their functional status depends on itsability to induce persistent increases in the activity of MAP kinase andthe signaling pathways in which it participates (Marshall (1995) Cell80:179-185). We and others (York et al. (1998) Nature 392:622-626) haveshown that NGF's ability to persistently activate MAP kinase is due toits ability to persistently activate an the small GTPase, called Rap1,that is upstream from MAP kinase. Using PC12 cells, neuron-like cellsthat have been used to model the signaling pathways activated by NGF andother neurotrophic factors, we asked whether estrogen would alsoactivate Rap1.

The first goal of these studies was to determine whether or not GPR30 isexpressed in PC12 cells. Using Northern blot and RT-PCR analyses, wefound that it was expressed (FIG. 1). In recent experiments, Westernblotting has shown that GPR30 protein is also present. Interestingly,GPR30 appears be the only estrogen receptor expressed in PC12 cells.Using the same methods, we did not detect the classical ERs in thesecells.

Once the expression of GPR30 in PC12 cells was confirmed, we set out toexamine estrogen effects on Rap1 signaling. We found that 17β-estradiolactivated Rap1, but not Ras (FIG. 2 A and B). Remarkably, it did so witha temporal pattern quite similar to what is seen with NGF. Like NGF,17β-estradiol had little effect on the activation of Rap1 during thefirst 10 minutes of treatment and had a marked effect at later times.While tamoxifen, an estrogen antagonist, alone had no effect on Rap1activity, it blocked the response to 17β-estradiol (FIGS. 2 C and D).This suggests that the ligand binding site in GPR30 for 17β-estradiolaccepts tamoxifen but is not activated by it. Importantly, like NGF,17β-estradiol induced sustained activation of MAP kinase (FIG. 3).Indeed, the pattern for activation of MAP kinase (labelled as pERK inthe Figure) closely paralleled that for activation of Rap1, a findingthat is also seen with NGF.

To ask if the signaling events induced by 17β-estradiol through GPR30were reflected in changes in the structure and function of neurons, wetested the effects of 17β-estradiol on neuronal differentiation andsurvival. We discovered that 17β-estradiol induced the outgrowth of longneurites from PC12 cells (FIG. 4). This effect mirrors the actions ofNGF. Like NGF, 17β-estradiol prevented these cells from dying underserum free culture, a condition well known to cause their death.Consistent with these findings, 17β-estradiol increased thedifferentiated state of PC12 cells by increasing the level of expressionof the 145 kDa intermediate neurofilament protein (FIG. 5). Theseobservations show that 17β-estradiol acts as a neurotrophic factor forPC12 cells that express GPR30 in the absence of other estrogenreceptors. They raise the possibility that estrogen signaling throughGPR30 has potent effects on the viability and function of neurons,including those whose survival is threatened.

Our observations provide strong support for the view that GPR30 mediatesthe neurotrophic actions of estrogen in neuronal cells. The focus onGPR30 as a neurotrophic receptor will potentially lead to important newinsights into estrogen signaling. This receptor is an important targetof future drug discovery efforts aimed at preventing and treatingneurodegenerative disorders.

What is claimed is:
 1. A method of increasing neurotrophic activity in aneural cell, the method comprising: administering an effective dose of aGPR30 ligand that activates GPR30 receptor, but not ESR1 or ESR2, to aneural cell expressing said GPR30 receptor, wherein activation of saidGPR30 receptor increases neurotrophic activity in said neural cell. 2.The method of claim 1, wherein said GPR30 ligand is an estrogenderivative.
 3. The method of claim 1, wherein said GPR30 ligand is aderivative of a non-steroidal estrogen.
 4. The method of claim 1,wherein said GPR30 ligand activates Rap1.
 5. The method of claim 1,wherein said GPR30 ligand activates MAP kinase.